Regulation of cytokine production in a hematopoietic cell

ABSTRACT

A method useful for regulating cytokine production by a hematopoietic cell by regulating an MEKK/JNKK-contingent signal transduction pathway in such a cell is disclosed. Methods of identifying compounds capable of specifically regulating an MEKK/JNKK-contingent signal transduction pathway in hematopoietic cells, a kit for identifying cytokine regulators, methods to treat diseases involving cytokine production, and cells useful in such methods are also set forth.

GOVERNMENT RIGHTS

This invention was made in part with government support under: AIHL-36577 and DK-37871, each awarded by the National Institutes ofHealth. The government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to a method for regulating anMEKK/JNKK-contingent signal transduction pathway in a hematopoietic cellin order to regulate cytokine production by such cell.

BACKGROUND OF THE INVENTION

Aggregation of the high-affinity Fc receptors for immunoglobulin E (IgE)(FcεRI) on the surface of mast cells initiates intracellular signaltransduction pathways, involving the tyrosine phosphorylation ofcellular proteins, activation of phospholipase Cγ, hydrolysis ofphosphoinositide, increase in intracellular calcium, activation ofprotein kinase C and the stimulation of phosphatidylinositol 3-kinase.These signal transduction pathways are believed to be involved in theexocytic release of inflammatory mediators such as vasoactive amines,cytokines, and lipid metabolites. The production of cytokines by mastcells is a critical event that influences the pathogenesis of allergicinflammation in asthma and other allergic disorders.

In addition to the activation of phospholipase Cγ and protein kinase C,which appears to be essential for the FcεRI-mediated release ofpreformed mediators, the aggregation of FcεRI on rat basophilic leukemia2H3 (RBL-2H3) cells has been shown to induce histamine and leukotrienerelease. Except for the activation of the extracellular signal-regulatedkinases/mitogen activated protein kinases (ERKs/MAPKs), however, thedownstream consequences of early activation events in a signaltransduction pathway leading to cytokine production are not welldefined.

The extracellular signal-regulated kinases (ERKs), ERK1 and ERK2, areserine/threonine protein kinases that are activated through concomitantphosphorylation of tyrosine and threonine residues. Prior to the currentinvention, it was thought that ERKs were one of the intermediates in thesignal transduction pathway leading to increases in gene transcriptionand proliferation, including cytokine gene transcription. ERKsphosphorylate specific transcription factors including members of theEts family, such as Elk-1, and it has been reported that ERKs areactivated via FcεRI on mast cells.

Despite the current understanding of early signal transduction events inhematopoietic cells, there remains a need to elucidate signaltransduction pathways that specifically regulate cytokine production insuch cells and to determine what molecules and/or functional elements ofsuch molecules are responsible for regulating such cellular pathways.There is also a need for products and processes that permit theeffective regulation of specific steps in such a signal transductionpathway. Regulation of specific steps of a signal transduction pathwaywhich regulate cytokine production permits the implementation ofpredictable controls of such signal transduction in cells, therebyallowing modulation of the effects of cytokine production in diseaseswherein such modulation can ameliorate disease pathogenesis.

SUMMARY OF THE INVENTION

The present invention generally relates to a method to regulate a novelsignal transduction pathway to modulate the production of cytokines by ahematopoietic cell. The present inventors have identified anMEKK/JNKK-contingent signal transduction pathway which regulates theproduction of cytokines by a hematopoietic cell. Prior to the presentinvention, it was thought that signal transduction through the ERKpathway lead to increases in gene transcription and proliferation,including cytokine gene transcription. The ERK pathway is known to bedistinct from the pathway of the present invention; therefore, thediscovery that an ERK-independent signal transduction pathway regulatescytokine production is unexpected. The present inventors were the firstto appreciate that an MEKK/JNKK-contingent signal transduction pathway,and not an ERK-dependent pathway, regulates cytokine production.Furthermore, the present inventors were the first to appreciate thatsuch an MEKK/JNKK-contingent pathway is activated in mast cells throughaggregation of FceRI and activation of PI3-kinase (PI3-K). The presentinventors were also the first to appreciate the method of regulation ofsuch a signal transduction pathway in order to regulate production ofcytokines such as, TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF,GM-CSF, TNF-b, TGF-b, IFN-γ, and IFN-α/β, in a hematopoietic cell suchas a mast cell, a basophil, an eosinophil, a neutrophil, a T cell, a Bcell, a macrophage, a dendritic cell, and a natural killer cell.

One embodiment of the present invention relates to a method to regulatecytokine production by regulating an MEKK/JNKK-contingent signaltransduction pathway in a hematopoietic cell. Preferably, such a methodcomprises regulating one or more of the signal transduction moleculeselected from the group of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 andJNK2.

In one embodiment, an MEKK/JNKK-contingent signal transduction pathwaycan be regulated by administration of a compound which regulates asignal transduction molecule selected from the group of MEKK1, MEKK2,MEKK3, MEKK4, JNKK, JNK1 and JNK2, such that cytokine production isregulated. Preferably, such a compound regulates such a signaltransduction molecule by a method such as degrading the molecule,binding an inhibitory compound to the molecule, inhibiting transcriptionof the molecule, inhibiting translation of the molecule, and inhibitingthe interaction of the molecule with another signal transductionmolecule.

A preferred embodiment of the present invention relates to a method toregulate cytokine production in a hematopoietic cell expressing FceRI byregulating an MEKK/JNKK-contingent signal transduction pathway in suchcell. Regulation of an MEKK/JNKK-contingent signal transduction pathwaycan further comprise regulating other signal transduction pathways thataffect the MEKK/JNKK-contingent signal transduction pathway.

Another embodiment of the present invention relates to a method toidentify compounds which regulate cytokine production in a hematopoieticcell. Such a method comprises contacting a cell with a putativeregulatory compound and determining whether such a compound is capableof regulating cytokine production in a cell by regulating anMEKK/JNKK-contingent signal transduction pathway in the cell.

Yet another embodiment of the present invention relates to a kit foridentifying compounds which regulate cytokine production by regulatingan MEKK/JNKK-contingent signal transduction pathway.

Another embodiment of the present invention relates to a method to treata disease involving cytokine production in an animal by regulating anMEKK/JNKK-contingent signal transduction pathway. In one embodiment,such a treatment involves administering to an animal an effective amountof a compound which interacts with a signal transduction molecule in anMEKK/JNKK-contingent signal transduction pathway such that cytokineproduction is regulated.

A preferred embodiment of the present invention relates to a method totreat allergic inflammation by regulating cytokine production. Suchregulation of cytokine production is effected by regulation of anMEKK/JNKK-contingent signal transduction pathway.

Yet another embodiment of the present invention relates to a compoundfor regulating cytokine production. Such a compound interacts with asignal transduction molecule in an MEKK/JNKK-contingent signaltransduction pathway in a manner effective to regulate cytokineproduction.

Another embodiment of the present invention relates to a cell used in amethod to identify compounds capable of regulating cytokine production,comprising a cell having at least one heterologous mammalian nucleicacid sequence encoding at least one protein involved in anMEKK/JNKK-contingent signal transduction pathway. A preferred embodimentrelates to a method of using such a cell to screen putative regulatorycompounds for their ability to regulate cytokine production in saidcell.

DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates an MEKK/JNKK-contingent signaltransduction pathway of the present invention.

FIG. 2A demonstrates activation of JNK by OVA and OVA-IgE in passivelysensitized mast cells.

FIG. 2B shows an immunoblot demonstrating JNK activity measured inpassively sensitized mast cells over time.

FIG. 2C graphically shows fold-increases in JNK activity in passivelysensitized mast cells.

FIG. 2D shows an immunoblot demonstrating the antigen-specificity of JNKactivation in passively sensitized mast cells.

FIG. 3A shows an immunoblot demonstrating the activation of MEKK1 byantigen in passively sensitized mast cells.

FIG. 3B graphically shows fold-increases in MEKK1 activation by antigenin passively sensitized mast cells.

FIG. 4A shows an immunoblot demonstrating the activation of ERK2 byantigen in passively sensitized mast cells.

FIG. 4B shows an immunoblot demonstrating ERK2 phosphorylation measuredin passively sensitized mast cells over time.

FIG. 4C shows an immunoblot demonstrating ERK2 kinase activity measuredin passively sensitized mast cells for up to 90 minutes.

FIG. 5A shows an immunoblot demonstrating that wortmannin inhibits JNKactivation by antigen in mast cells.

FIG. 5B demonstrates that a decrease in JNK activity correlates withincreased wortmannin concentration.

FIG. 5C shows that wortmannin does not inhibit ERK2 activity.

FIG. 6 demonstrates that TNF-a is produced in response to antigen bypassively sensitized mast cells.

FIG. 7 shows the fold-increase in TNF-a production over time in responseto antigen activation of passively sensitized mast cells.

FIG. 8 demonstrates that p38 MAP kinase is activated in response toantigen activation of passively sensitized mast cells.

FIG. 9 shows that wortmannin inhibits p38 MAP kinase activation inactivated MC/9 cells.

FIG. 10 demonstrates that wortmannin inhibits TNF-a production byactivated MC/9 mast cells.

FIG. 11 shows that an MEK inhibitor, PD 098059, inhibits activation ofERK2 in activated MC/9 cells.

FIG. 12 shows that MEK inhibitor, PD 098059, does not inhibit TNF-αproduction in MC/9 cells.

FIG. 13 demonstrates that the PI3-K inhibitor, wortmannin, inhibitsTNF-A promoter activity in MC/9 cells stimulated by FcεRI aggregation.

FIG. 14 shows that MEK inhibitor, PD 098059, enhances TNF-α promoteractivity in MC/9 cells stimulated by FcεRI aggregation.

FIG. 15 demonstrates that overexpression of MEKK1 greatly enhances JNKactivity in antigen-activated MC/9 cells.

FIG. 16 shows that overexpression of MEKK1 weakly enhances p38 activityin antigen-activated MC/9 cells.

FIG. 17 demonstrates that overexpression of MEKK1 greatly enhances TNF-αpromoter activity in antigen-activated MC/9 cells. FIG. 18 demonstratesthat cross-linking of c-kit on MC/9 cells synergizes with aggregation ofFceRI to greatly enhance JNK activation.

FIG. 19 demonstrates that cross-linking of c-kit on MC/9 cellssynergizes with aggregation of FceRI to greatly enhance TNF-aproduction.

FIG. 20 shows that aggregation of FceRI on MC/9 cells activates thetranscription factor, NFkB.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the elucidation of a novel signaltransduction pathway that regulates cytokine production in hematopoieticcells and a method to target such a pathway to regulate cytokineproduction by such cells. The regulation of cytokine production in ahematopoietic cell is useful since cytokines are known to play acritical role in the pathogenesis of many diseases. In particular,regulation of cytokine production in a cell expressing FceRI, such as amast cell, is useful for treating diseases involving allergicinflammation. Specifically, the present invention relates to theregulation of an MEKK/JNKK-contingent signal transduction pathway, inorder to regulate cytokine production in a hematopoietic cell.

Prior to the present invention, it was thought that signal transductionthrough the ERK pathway lead to increases in gene transcription andproliferation, including cytokine gene transcription. Since the ERKpathway is known to be distinct from the pathway of the presentinvention, the present discovery that an ERK-independent signaltransduction pathway regulates cytokine production is unexpected andsurprising.

As used herein, the phrase "signal transduction pathway" refers to atleast one biochemical reaction, but more commonly a series ofbiochemical reactions, which result from interaction of a cell with astimulatory compound. Thus, the interaction of a stimulatory compoundwith a cell generates a "signal" that is transmitted through the signaltransduction pathway, ultimately resulting in a cellular response.

A signal transduction pathway of the present invention can include avariety of signal transduction molecules that play a role in thetransmission of a signal from one portion of a cell to another portionof a cell. Signal transduction molecules of the present inventioninclude, for example, cell surface receptors and intracellular signaltransduction molecules. As used herein, the phrase "cell surfacereceptor" includes molecules and complexes of molecules capable ofreceiving a signal and the transmission of such a signal across theplasma membrane of a cell. An example of a "cell surface receptor" ofthe present invention is FceRI. As used herein, the phrase"intracellular signal transduction molecule" includes those molecules orcomplexes of molecules involved in transmitting a signal from the plasmamembrane of a cell through the cytoplasm of the cell, and in someinstances, into the cell's nucleus. In the present invention, MEKK1,MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2 are "intracellular signaltransduction molecules", but can also be referred to as "signaltransduction molecules".

A signal transduction pathway in a cell can be initiated by interactionof a cell with a stimulator that is inside or outside of the cell. If anexterior (i.e. outside of the cell) stimulator interacts with a cellsurface receptor, a signal transduction pathway can transmit a signalacross the cell's membrane, through the cytoplasm of the cell, and insome instances into the nucleus. If an interior (e.g. inside the cell)stimulator interacts with an intracellular signal transduction molecule,a signal transduction pathway can result in transmission of a signalthrough the cell's cytoplasm, and in some instances into the cell'snucleus.

As used herein, the term "molecule" refers to a protein, a lipid, anucleic acid or an ion, and at times is used interchangeably with suchterms. In particular, a signal transduction molecule refers to aprotein, a lipid, a nucleotide, or an ion involved in a signaltransduction pathway.

A signal transduction molecule of the present invention can regulate theactivity of proteins involved in the transcription of genes involved incell growth within the nucleus of a cell, in particular, cytokine genes,thereby altering the biological function of a cell.

Signal transduction can occur through: the phosphorylation of amolecule; non-covalent allosteric interactions; complexing of molecules;the conformational change of a molecule; calcium release; inositolphosphate production; proteolytic cleavage; cyclic nucleotide productionand diacylglyceride production. Preferably, signal transduction occursthrough phosphorylating a signal transduction molecule including, MEKK1,MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2. According to the presentinvention, all the signal transduction molecules of theMEKK/JNKK-contingent signal transduction pathway need not be known inorder to successfully utilize the methods of the present invention.

According to the present invention, an MEKK/JNKK-contingent signaltransduction pathway refers generally to a pathway in which MEKK proteinregulates a pathway that includes JNKK, molecules which are activebetween MEKK and JNKK in the pathway, and molecules downstream of JNKK,such as JNK1, JNK2, NF-AT, AP-1, Jun, Fos, ATF-2, NFkB, and CBP. AnMEKK/JNKK-contingent signal transduction pathway is independent of anERK-dependent signal transduction pathway downstream from the effects ofMEKK. In other words, the regulation of the MEKK/JNKK-contingent signaltransduction pathway does not, of necessity, also affect signaltransduction downstream from the ERK protein. A suitableMEKK/JNKK-contingent signal transduction pathway includes a pathwayinvolving an MEKK\JNKK-contingent signal transduction molecule,including MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2, but not ERKmolecules exclusively involved in an ERK-dependent signal transductionpathway. More particularly, an MEKK\JNKK-contingent molecule regulates apathway that is substantially independent of an ERK-dependent pathway ifthe MEKK/JNKK-contingent protein induces phosphorylation of JNKK or amember of the pathway downstream of JNKK (e.g., proteins including JNK1,JNK2, NF-AT, AP-1, Jun, Fos, ATF-2, NFkB, and/or CBP), and inducescytokine production in a cell having such a pathway. A schematicrepresentation of the proposed signal transduction pathway of thepresent invention is shown in FIG. 1.

As a result of the elucidation by the present inventors of the novelfunction of MEKK/JNKK-contingent signal transduction pathway (i.e.regulation of cytokine production), one of skill in the art candetermine that regulation of such a pathway by an MEKK/JNKK-contingentmolecule is substantially independent of an ERK-pathway. One can alsodetermine how an MEKK/JNKK-contingent molecule regulates thephosphorylation of a downstream member of an MEKK/JNKK-contingentpathway or, alternatively, how to regulate cytokine production in a cellthrough the inhibition and/or stimulation of the MEKK/JNKK-contingentpathway.

An "ERK-dependent pathway" can refer to a signal transduction pathway inwhich ERK protein regulates a signal transduction pathway that issubstantially independent of an MEKK/JNKK-contingent pathway, or apathway in which ERK protein regulation converges with common members ofa pathway involving MEKK/JNKK-contingent protein. More particularly, anERK-dependent pathway includes components downstream of ERK proteins andcontinues downstream in a series of signal transduction events. Theindependence of regulation of a pathway by an ERK protein from theregulation of a pathway by an MEKK/JNKK-contingent protein can bedetermined using methods as described above in relation to theMEKK/JNKK-contingent pathway. In particular, regulation of anERK-dependent pathway will not lead to the regulation of cytokineproduction in a cell.

As referenced in the present invention, MEKK, or mitogen ERK kinasekinase, is a signal transduction molecule that is capable ofphosphorylating mitogen ERK kinase or MAPK kinase (MEK) and/or c-Junamino-terminal kinase kinase (JNKK), thereby activating such molecules.Several members of the MEKK family have been identified, includingMEKK1, MEKK2, MEKK3, and MEKK4. An MEKK molecule of theMEKK/JNKK-contingent signal transduction pathway of the presentinvention phosphorylates JNKK. In a preferred embodiment, MEKK1phosphorylates JNKK in response to activation through anFceRI-activated, PI3-kinase-dependent pathway in a mast cell. MEKprotein is not a component of the MEKK/JNKK-contingent signaltransduction pathway of the present invention.

JNK-activating protein kinase (JNKK), is a dual-specificitythreonine-tyrosine protein kinase that activates JNK and functionsdownstream from MEKK. JNK is a distant member of the mitogen-activatedprotein kinase superfamily, designated c-Jun amino-terminal kinase(JNK). JNK is activated following dual phosphorylation at a Thr-Pro-Tyrmotif in response to diverse stimuli including tumor necrosis factor-α,heat shock, or ultraviolet irradiation. Costimulation of T cells withantibodies to the T cell receptor and CD28 or the stimulation of B cellswith anti-CD40 antibody also induces the activation of JNK. JNKfunctions to phosphorylate c-Jun at the amino-terminal regulatory sites,serine 63 and serine 73, mapping within its transactivation domain.Phosphorylation of these sites in response to UV irradiation alsoresults in the transcriptional activation of c-Jun. There are twomembers of the JNK family, designated JNK1 and JNK2. It has beensuggested that JNK may be involved in apoptosis, but until the presentinvention, it was not appreciated that JNK was involved in a signaltransduction pathway that regulated cytokine production in a cell.

One embodiment of the present invention is directed to a method toregulate cytokine production in a hematopoietic cell, comprisingregulating an MEKK/JNKK-contingent signal transduction pathway. In apreferred embodiment, regulation of such a pathway results in inhibitionof cytokine production.

As used herein, the term "regulate" can be used interchangeably with theterm "modulate". "To regulate" a molecule, a pathway, or a function ofsuch a molecule or pathway, in the present invention refers tospecifically controlling, or influencing the activity of such amolecule, pathway, or function, and can include regulation byactivation, stimulation, inhibition, alteration or modification of suchmolecule, pathway or function.

In a preferred embodiment, regulating an MEKK/JNKK-contingent signaltransduction pathway comprises regulating a signal transduction moleculeselected from the group consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK,JNK1, and JNK2. Preferably, regulation of such a signal transductionmolecule is accomplished by a method including, but not limited to,degrading said molecule, binding a regulatory compound to said molecule,inhibiting transcription of said molecule, inhibiting translation ofsaid molecule, inhibiting activation of said molecule, and inhibitingthe interaction of said molecule with another signal transductionmolecule.

In a preferred embodiment of the present invention, anMEKK/JNKK-contingent signal transduction pathway is regulated byadministration of an effective amount of a compound that interacts witha signal transduction molecule of said pathway such that cytokineproduction is regulated. Preferably, such a compound regulates a signaltransduction molecule selected from the group of MEKK1, MEKK2, MEKK3,MEKK4, JNKK, JNK1 and JNK2. In another embodiment, such a compoundregulates PI3-K. A regulatory compound of the present invention,however, does not regulate a molecule specific to an ERK-dependentpathway. Thus, although in some cell types, for example, PI3-K mayregulate other signal transduction pathways in addition to anMEKK/JNKK-contingent pathway, regulation of PI3-K to effect regulationof an MEKK/JNKK-contingent pathway is still within the scope of thepresent invention.

As used herein, an "effective amount" of a compound is at least theminimum amount of a compound that is necessary to minimally achieve, andmore preferably, optimally achieve, the desired effect (i.e. regulationof a signal transduction molecule). An effective amount for use in agiven method can be readily determined by one skilled in the art withoutundue experimentation, depending upon the particular circumstancesencountered (e.g. concentrations, cell type and number, etc.).

A regulatory compound of the present invention regulates cytokineproduction in a hematopoietic cell, comprising a compound that iscapable of regulating an MEKK/JNKK-contingent signal transductionpathway of the present invention. Such a regulatory compound includes acompound that is capable of inhibiting an MEKK/JNKK-contingent signaltransduction pathway of the present invention, a compound that iscapable of stimulating an MEKK/JNKK-contingent signal transductionpathway of the present invention, or a compound that is capable ofpreventing both the stimulation and the inhibition of the activity of anMEKK/JNKK-contingent signal transduction pathway of the presentinvention (i.e., maintaining the activity of a signal transductionpathway). Such regulation by a compound can be effected by, but is notlimited to, any of the preferred methods of regulating a signaltransduction molecule as described above (i.e. degrading a signaltransduction molecule, etc.).

Acceptable protocols to contact a cell with a regulatory compound in aneffective manner can be accomplished by those skilled in the art basedon variables such as, the conditions under which the compound is beingadministered, the type of cell being regulated and the chemicalcomposition of the regulatory compound (i.e., size, charge etc.) beingadministered.

As used herein, "inhibiting the interaction of" one molecule withanother can be accomplished in a variety of ways including, but notlimited to, physically blocking the interaction between two molecules(i.e. by a regulatory compound), moving one molecule relative to theother such that interaction between the two can not occur,dephosphorylating or preventing phosphorylation of one or both moleculessuch that interaction can not occur, and phosphorylating one or bothmolecules such that interaction can not occur.

Inhibiting activation of a molecule can be accomplished by a methodincluding, but not limited to, preventing activation of said moleculeand deactivating a molecule that is activated. Such methods include, butare not limited to, phosphorylating a molecule, dephosphorylating amolecule, preventing phosphorylation of a molecule, physicallyinhibiting activation of a molecule as described above, and degrading amolecule.

In one embodiment of the present invention, signal transduction pathwaysinvolved in cytokine production in a hematopoietic cell are regulated bya method comprising inhibiting the interactions between moleculesselected from a group of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 andJNK2, in a hematopoietic cell having an MEKK/JNKK-contingent signaltransduction pathway. Such inhibition of interactions between suchsignal transduction molecules can be effected by methods of regulationdescribed herein, including, but not limited to, contacting a cell witha compound which modulates the interactions between such molecules.

According to the present invention, a hematopoietic cell is a cell whichincludes erythrocyte cells (i.e., a red blood cell), certain leukocytecells, including granular leukocytes (eosinophils, basophils,neutrophils, and mast cells), non-granular leukocytes (megakaryocytes,polymorphonuclear cells, lymphocytes and monocytes), or thrombocytecells (i.e., platelet cell). A preferred hematopoietic cell of thepresent invention includes a mast cell, a basophil, an eosinophil, aneutrophil, a T cell, a B cell, a macrophage, a dendritic cell, and anatural killer cell.

Cytokine production, as used in the present invention, refers to the denovo synthesis of mRNA encoding such a cytokine which results in thetranslation and exocytosis of such a cytokine. A cytokine for whichproduction can be regulated in the present invention can be selectedfrom the group consisting of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,G-CSF, GM-CSF, TNF-b, TGF-b, IFN-γ, and IFN-α/β. It is within the scopeof the present invention that cytokines that are unknown at the time ofthe present invention, but are described in the art in the future, mayalso be a cytokine that can be regulated by the MEKK/JNKK-contingentpathway of the present invention. In a preferred embodiment, productionof TNF-a by a mast cell is regulated. The de novo synthesis of cytokineproduction is regulated by activation of an MEKK/JNKK-contingent signaltransduction pathway of the present invention. In a preferredembodiment, activation of such a pathway results in modulation of theinteraction of transcription factors with cytokine promoters which canthereby regulate cytokine production.

A cytokine promoter as described herein can include any DNA sequencecapable of being specifically bound by an RNA polymerase in such amanner that the RNA polymerase can unwind the DNA strand to initiate RNAsynthesis of a cytokine gene.

A transcription factor of the present invention is capable of mediatingthe rate of cytokine transcription in a cell. The rate of transcriptionof a particular cytokine DNA molecule in a cell is not necessarily fixedand can change according to the needs of the cell in differentconditions of growth. Such regulation of transcription can be mediatedby proteins that, by binding to DNA near or within a promoter, canincrease or decrease the rate at which RNA polymerase initiates RNAsynthesis. Transcription rates can be mediated by proteins includingtranscription factors. Suitable transcription factors include, but arenot limited to, at least a portion of a transcription factor. Atranscription factor as referred to herein has a transactivation domaincapable of regulating the activity of the transcription factor. Such atranscriptional activation domain contains amino acid residues capableof being phosphorylated, the phosphorylation of which results in theregulation of the activity of the transcription factor. Without beingbound by theory, it is believed that phosphorylation of residuescontained in a transcriptional activation domain alters the conformationof the transcription factor such that the DNA binding domain of thetranscription complex can drive transcription. Preferred sites ofphosphorylation include serine residues and threonine residues spaced insuch a manner that the phosphorylation of such residues results in theactivation of the transcription factor. Preferred transcription factorsof the present invention include NF-AT, Ap-1, Jun, Fos, ATF-2, NFkB, andCBP.

In one embodiment of the present invention, a hematopoietic cell, inwhich cytokine production is regulated, expresses FceRI. Preferably,cytokine production in such a cell is inhibited by regulation of anMEKK/JNKK-contingent signal transduction pathway. In a preferredembodiment, activation of an MEKK/JNKK-contingent signal transductionpathway is initiated through aggregation of FceRI. FceRI is thehigh-affinity receptor for IgE. Hematopoietic cells expressing FceRI inthe present invention are preferably mast cells, basophils andeosinophils, and most preferably mast cells. The multivalent binding ofan antigen to receptor-bound IgE and the subsequent aggregation of thehigh-affinity Fc receptors for IgE (FcεRI) provide the trigger foractivation of mast cells. The first demonstrable response to FcεRIaggregation is tyrosine phosphorylation and activation of phospholipaseCγ, which catalyzes the hydrolysis of phosphatidylinositol4,5-bisphosphate resulting in the liberation of inositol1,4,5-trisphosphate and diacylglycerol. The elevation of diacylglyceroland the mobilization of Ca²⁺ from intracellular and extracellularsources results in the activation of protein kinase c.

The FcεRI is composed of three subunits, single α and β chains, and ahomodimer of disulfide-linked γ chains. The intracellular tails of the βand γ chains contain a motif that is important for signal transduction.This motif has been called the antigen recognition activation motif ortyrosine activation motif, which is thought to couple the FcεRI toprotein tyrosine kinases. Activation of protein tyrosine kinases is oneof the earliest signaling events induced by aggregation of the FceRI onmast cells. The aggregation of FcεRI initiates diverse signaltransduction pathways. As is shown for the first time herein, one ofthese pathways is the MEKK/JNKK-contingent signal transduction pathwaywhich leads to cytokine production.

In a preferred embodiment of the present invention, anMEKK/JNKK-contingent signal transduction pathway is activated through aphosphatidylinositol 3-kinase (PI3-K) signal transduction pathway. Moreparticularly, such a PI3-K signal transduction pathway is activatedthrough aggregation of FceRI on the surface of a mast cell, a basophil,or an eosinophil. As used herein, a PI3-K pathway is a signaltransduction pathway that involves the signal transduction molecule,PI3-K. PI3-K is a heterodimeric protein composed of a non-catalytic p85α subunit (85 kD) and a catalytic p110 β subunit (110 kD). PI3-Kγ is a110 kD enzyme specifically regulated by G proteins. PI3-K is capable ofphosphorylating inositol lipids on the D-3 hydroxyl position. Containedwithin the p85 subunit are two proline-rich domains. PI3-Kγ is reguledby G protein subunits, particularly βγ subunits.

The present inventors have unexpectedly found that the PI3-K inhibitor,wortmannin, at concentrations that inhibit PI3-kinase activity, alsoinhibited JNK activation, but not ERK activation. This finding is thefirst demonstration of a role for PI3-kinase in regulating a JNK pathwayby an Src family tyrosine kinase-associated receptor. Thus, in mastcells the regulation of the MEKK1, JNKK, JNK pathway is dependent on theactivation of PI3-kinase, which in turn, is activated by aggregation ofFceRI. Mechanistically, there is a very early separation in the signalpathways activated by the FcεRI to differentially regulate JNK and ERKsequential protein kinase pathways. Without being bound by theory, thepresent inventors believe that PI3-kinase activity is involved inactivating the MEKK/JNKK-contigent pathway in mast cells downstream oftyrosine kinases and upstream of MEKK1.

In a preferred embodiment of the present invention, regulation ofcytokine production by regulating an MEKK/JNKK-contingent signaltransduction pathway further comprises regulating PI3-kinase.

Such regulation can be effected by the methods described herein forregulation of an MEKK/JNKK-contingent signal transduction molecule,and/or by interfering with the capability of PI3-K to trigger downstreamsignal transduction events. In yet another preferred embodiment of thepresent invention, a method to regulate cytokine production in a cellfurther comprises regulating a signal transduction pathway selected fromthe group of a c-kit signal transduction pathway and a p38 signaltransduction pathway. The c-kit and the p38 signal transduction pathwaysare distinct from the MEKK/JNKK-contingent signal transduction pathwayof the present invention. It is appreciated for the first time in thepresent invention, that both the c-kit and the p38 signal transductionpathways can enhance the regulation of cytokine production that iseffected by the MEKK/JNKK-contingent signal transduction pathway. c-kitis a cell-surface receptor that, when bound by c-kit ligand, initiates asignal transduction pathway that is incapable of regulating cytokineproduction itself, but that can enhance the effects of theMEKK/JNKK-contingent pathway on cytokine production. p38 is activated bydual phosphorylation at a Thr-Gly-Tyr motif and is activated by cellularstress, pro-inflammatory cytokines, and lipopolysaccharide (LPS). p38can be activated by a specific JNKK referred to as MKK3 or MKK6;however, in the MEKK/JNKK-contingent signal transduction pathway of thepresent invention, JNK is preferentially activated over p38. Therefore,signal transduction through the MEKK/JNKK-contingent signal transductionpathway is primarily responsible for regulation of cytokine productionin a cell, but can be enhanced by other signal transduction pathways.Regulation of such other pathways can be effected by the methodsdescribed herein for regulation of an MEKK/JNKK-contingent signaltransduction pathway.

One aspect of the present invention includes a method to identifycompounds that regulate an MEKK/JNKK-contingent signal transductionpathway. Such compounds are referred to herein as "putative regulatorycompounds". As used herein, the term "putative" refers to compoundshaving an unknown signal transduction regulatory activity, at least withrespect to the ability of such compounds to regulate cytokine productionvia the MEKK/JNKK-contingent pathway. Regulatory compounds, defined bytheir identifying characteristics of being capable of regulating signaltransduction molecules of the present invention have been previouslydescribed herein.

Putative regulatory compounds as referred to herein include, forexample, compounds that are products of rational drug design, naturalproducts and compounds having partially defined signal transductionregulatory properties. A putative compound can be a protein-basedcompound, a carbohydrate-based compound, a lipid-based compound, anucleic acid-based compound, a natural organic compound, a syntheticallyderived organic compound, an anti-idiotypic antibody and/or catalyticantibody, or fragments thereof. A putative regulatory compound can beobtained, for example, from libraries of natural or synthetic compounds,in particular from chemical or combinatorial libraries (i.e., librariesof compounds that differ in sequence or size but that have the samebuilding blocks) or by rational drug design.

A method to identify a regulatory compound of the present inventioncomprises the steps of providing a hematopoietic cell having anMEKK/JNKK-contingent signal transduction pathway, contacting such a cellwith a putative regulatory compound, and determining whether such acompound is capable of regulating said MEKK/JNKK-contingent signaltransduction pathway. In a preferred embodiment, such a method is usedto identify a regulatory compound that inhibits cytokine production bysaid cell.

In particular, a preferred regulatory compound of the present inventioncan be identified by determining the ability of such a compound tomodulate the interactions between signal transduction molecules selectedfrom the group of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2. Morepreferably, the regulation of such a signal transduction molecule by aregulatory compound of the present invention modulates the interactionof such a molecule with a transcription factor selected from the groupof NF-AT, Ap-1, Jun, Fos, ATF-2, NFkB and CBP. In another preferredembodiment, a regulatory compound is identified by its ability tomodulate the interaction of PI3-K with signal transduction molecules inthe MEKK/JNKK-contingent pathway.

Another embodiment of the present invention is a kit for identifyingcompounds capable of regulating cytokine production in a hematopoieticcell. Such a kit comprises a hematopoietic cell capable of producing anamount of at least one cytokine, such cell having anMEKK/JNKK-contingent signal transduction pathway, and production of suchcytokine being dependent on such MEKK/JNKK-contingent signaltransduction pathway. Such a kit further comprises a means fordetermining a change in said amount of cytokine produced by such a cellafter the cell is contacted with a putative regulatory compound. In apreferred embodiment, such a kit is useful for identifying compoundsthat inhibit cytokine production by said cell.

As used herein, "at least one cytokine" means that a minimum of onecytokine selected from the group of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,G-CSF, GM-CSF, TNF-b, TGF-b, IFN-γ, and IFN-α/β., can be produced by acell of the kit of the present invention. It is within the scope of thepresent invention that more than one cytokine can be produced by such acell. As used herein, "an amount" of a cytokine refers to a quantity ofcytokine produced by a hematopoietic cell that is detectable by standardmethods of cytokine measurement in the art. "An amount" can refer to adetectable protein concentration of cytokine in a sample, or to units ofactivity of such a cytokine in a sample. "A change in the amount" ofcytokine produced by a hematopoietic cell after such a cell is contactedby a putative regulatory compound, is any detectable change, (i.e.increase or decrease) in the amount of cytokine produced by such a cellafter contact with such a regulatory compound, as compared to the amountof cytokine produced before such cell was contacted with such acompound.

Suitable cells for use with either the method to detect a regulatorycompound or with the kit useful for detecting a regulatory compound ofthe present invention include any cell that has an MEKK/JNKK-contingentsignal transduction pathway. Such cells can include normal cells ortransformed derivatives thereof, that express a receptor in a nativephysiological context (e.g., basophils, eosinophils, neutrophils,monocytes, macrophages, and lymphoid cells). Alternatively, cells foruse with the present invention can include spontaneously occurringvariants of normal cells, or genetically engineered cells, that havealtered signal transduction activity, such as enhanced responses toparticular ligands. Signal transduction variants of normal cells can beidentified using methods known to those in the art. For example,variants can be selected using fluorescence activated cell sorting(FACS) based on the level of calcium mobilization by a cell in responseto a ligand. Genetically engineered cells can include recombinant cellsof the present invention (described in detail below) that have beentransformed with, for example, a recombinant molecule encoding a signaltransduction molecule and/or a transcription indicator recombinantmolecule of the present invention.

Cells for use with the present invention include mammalian,invertebrate, plant, insect, fungal, yeast and bacterial cells.Preferred cells include mammalian, amphibian and yeast cells. Preferredmammalian cells include primate, mouse and rat cells. In a preferredembodiment, cells to be used in a method to identify compounds whichregulate an MEKK/JNKK-contingent signal transduction pathway can begenetically manipulated to obtain cells having an MEKK/JNKK-contingentsignal transduction pathways wherein production of cytokines by suchcells is dependent on the MEKK/JNKK-contingent signal transductionpathway and/or components of such signal transduction pathway. In apreferred embodiment, such cells are substantially devoid of any othersignal transduction pathways that result in significant production ofcytokines by such cell.

In yet another embodiment, a cell suitable for use in the presentinvention further comprises a PI3-K signal transduction pathway whichactivates said MEKK/JNKK-contingent pathway, and/or a p38 signaltransduction pathway or a c-kit signal transduction pathway whichenhances said MEKK/JNKK-contingent pathway.

In one embodiment, a cell suitable for use in the present invention hasat least one type of cell surface receptor. A cell surface receptor asreferred to herein includes those cell surface receptors capable ofbinding to a ligand (as described in detail below) and capable ofinitiating an MEKK/JNKK-contingent signal transduction pathway in a cellupon ligand binding. A cell surface receptor typically includes anexternal portion located on the outer surface of a plasma membrane of acell, a transmembrane portion that spans the plasma membrane, and acytoplasmic portion located on the inner surface of the plasma membrane.

A cell surface receptor as described herein can be produced byexpression of a naturally occurring gene encoding a cell surfacereceptor and/or a heterologous nucleic acid molecule transformed into acell. An example of a cell surface receptor of the present inventioncould be, for example, CD40, CD28, or FceRI. A preferred cell surfacereceptor of the present invention is FceRI.

An intracellular signal transduction molecule as described herein can beproduced in a cell by expression of a naturally occurring gene and/or byexpression of a heterologous nucleic acid molecule transformed into thecell. Preferred intracellular signal transduction molecules of thepresent invention are MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2.MAPK/ERK molecules are not intracellular signal molecules of the presentinvention.

In certain embodiments, a cell of the present invention is transformedwith at least one heterologous nucleic acid sequence. A nucleic acidsequence, or molecule, as described herein can be DNA, RNA, or hybridsor derivatives of either DNA or RNA. Nucleic acid molecules as referredto herein can include regulatory regions that control expression of thenucleic acid molecule (e.g., transcription or translation controlregions), full-length or partial coding regions, and combinationsthereof. It is to be understood that any portion of a nucleic acidmolecule can be produced by: (1) isolating the molecule from its naturalmilieu; (2) using recombinant DNA technology (e.g., PCR amplification,cloning); or (3) using chemical synthesis methods. A gene includes allnucleic acid sequences related to a natural cell surface receptor genesuch as regulatory regions that control production of a cell surfacereceptor encoded by that gene (such as, but not limited to,transcription, translation or post-translation control regions) as wellas the coding region itself.

A nucleic acid molecule can include functional equivalents of naturalnucleic acid molecules encoding a protein or functional equivalents ofnatural nucleic acid sequences capable of being bound by proteins.Functional equivalents of natural nucleic acid molecules can include,but are not limited to, natural allelic variants and modified nucleicacid molecules in which nucleotides have been inserted, deleted,substituted, and/or inverted in such molecules without adverselyaffecting the function of products encoded by such sequences.

As guidance in determining what particular modifications can be made toany particular nucleic acid molecule, one of skill in the art shouldconsider several factors that, without the need for undueexperimentation, permit a skilled artisan to appreciate workableembodiments of the present invention. For example, such factors includemodifications to nucleic acid molecules done in a manner so as tomaintain particular functional regions of the encoded proteinsincluding, a ligand binding site, a target binding site, a catalyticdomain, etc. Functional tests for these various characteristics (e.g.,ligand binding studies and signal transduction assays such as kinaseassays, transcription assays, and other assays described in detailherein) allows one of skill in the art to determine what modificationsto nucleic acid sequences would be appropriate and which would not.

Transformation of a heterologous nucleic acid molecule (e.g., aheterologous cell surface receptor encoding nucleic acid molecule) intoa cell suitable for use in the present invention can be accomplished byany method by which a gene is inserted into a cell. Transformationtechniques include, but are not limited to, transfection, retroviralinfection, electroporation, lipofection, bacterial transfer andspheroplast fusion. Nucleic acid molecules transformed into cellssuitable for use in the present invention can either remain onextra-chromosomal vectors or can be integrated into the cell genome.

Expression of a nucleic acid molecule of the present invention in a cellcan be accomplished using techniques known to those skilled in the art.Briefly, the nucleic acid molecule is inserted into an expression vectorin such a manner that the nucleic acid molecule is operatively joined toa transcription control sequence in order to be capable of affectingeither constitutive or regulated expression of the gene when the gene istransformed into a host cell. The phrase "recombinant molecule", as usedherein refers to a gene operatively linked to at least one transcriptioncontrol sequence on an expression vector. The phrase "expressionvector", as used herein refers to a DNA or RNA vector that is capable oftransforming a host cell, of replicating within the host cell, and ofaffecting expression of the operatively linked gene. Expression vectorsare capable of replicating to either a high or low copy number dependingon their inherent characteristics. Transcription control sequences,which can control the amount of protein produced, include sequences thatcontrol the initiation, elongation, and termination of transcription.Particularly important transcription control sequences are those whichcontrol transcription initiation, such as promoter and upstreamactivation sequences.

Construction of desired expression vectors can be performed by methodsknown to those skilled in the art and expression can be in eukaryotic orprokaryotic systems. Procaryotic systems typically used are bacterialstrains including, but not limited to various strains of E. coli,various strains of bacilli or various species of Pseudomonas. Inprokaryotic systems, plasmids are used that contain replication sitesand control sequences derived from a species compatible with a hostcell. Control sequences can include, but are not limited to promoters,operators, enhancers, ribosome binding sites, and Shine-Dalgarnosequences. Expression systems useful in eukaryotic host cells comprisepromoters derived from appropriate eukaryotic genes. Useful mammalianpromoters include early and late promoters from SV40; other viralpromoters such as those derived from baculovirus, polyoma virus,adenovirus, bovine papilloma virus, avian sarcoma virus orcytomegalovirus; or collagenase promoters. Expression vectors includeany vectors that function (i.e., direct gene expression) in recombinantcells of the present invention including bacterial, yeast, other fungal,insect, and mammalian cells. Particularly preferred expression vectorsinclude promoters useful for expressing recombinant molecules in humancells.

An expression system can be constructed from any of the foregoingcontrol elements operatively linked to nucleic acid sequences usingmethods known to those of skill in the art. (see, for example, Sambrooket al., ibid.).

The conditions under which the cell of the present invention iscontacted with, such as by mixing, a putative regulatory compound areconditions in which the cell can transduce a normal signal ifessentially no regulatory compound is present. Such methods are withinthe skill in the art, and include an effective medium in which the cellcan be cultured such that the cell can exhibit signal transductionactivity. A preferred number of cells to use in the method or test kitof the present invention includes a number of cells that enables one todetect a change in activity of a signal transduction molecule using adetection method of the present invention (described in detail below).

In another embodiment of the present invention, cells suitable for usein the present invention are stimulated with ligands capable of bindingto cell surface receptors of the present invention to initiate anMEKK/JNKK-contingent signal transduction pathway and thereby regulatecytokine production. Suitable ligands can include, for example,hormones, growth factors, antigens, peptides, ions, otherdifferentiation agents and other cell type specific mitogens. Preferredligands include IgE, anti-FceRI, and c-kit ligand.

In another embodiment of the present invention, cells suitable for usein the present invention are stimulated with intracellular initiatormolecules capable of initiating a signal transduction pathway frominside a cell. Examples of intracellular initiator molecules as referredto herein include, but are not limited to, phorbol esters, calciumionophores, ALF4, phenyloxide, mastoparans, sodium orthovanadate,arachidonic acid and ceramides.

A suitable amount of putative regulatory compound(s) suspended inculture medium is added to the cells that is sufficient to regulate theactivity of a signal transduction molecule inside the cell such that theregulation is detectable using a detection method of the presentinvention. A preferred amount of putative regulatory compound(s)comprises between about 1 nM to about 10 mM of putative regulatorycompound(s) per well of a 96-well plate. The cells are allowed toincubate for a suitable length of time to allow the putative regulatorycompound to enter a cell and interact with a signal transductionmolecule. A preferred incubation time is between about 1 minute to about12 hours.

In one embodiment, the method and kit of the present invention includedetermining if a putative regulatory compound is capable of regulatingan MEKK/JNKK-contingent signal transduction pathway by regulatingcytokine production in a cell. Such methods of determining a change inan amount of a cytokine after contact of a putative regulatory compoundwith a cell include: immunoassays for cytokine production, such as byenzyme-linked immunoassay (e.g., ELISA), radioimmunoassay analysis,fluorescence immunoassay or immunoblot assay (as generally described inSambrook et al., ibid.); transcription assays to detect the activationof cytokine transcription, such as measuring the increase or decrease inmRNA transcription of a cytokine gene by PCR-based technology; andbiological assays in which a cytokine-indicator cell is used todetermine a change in an amount of cytokine (such cells are known in theart for a large number of cytokines). Particularly useful assays areantibody-based capture assays that comprise: (1) attaching a captureantibody having specificity for a specific cytokine to a support, suchas an ELISA plate; (2) contacting a cell supernate with thesubstrate-bound antibody to form an immune complex; (3) contacting thesubstrate-bound immune complex with a detection antibody specific for anepitope on the cytokine; and (4) detecting the association of thedetection antibody to the immune complex.

It is within the scope of the present invention to determine regulationof an MEKK/JNKK-contingent signal transduction pathway by measuring theactivation of signal transduction molecules selected from the group ofMEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2, by methods such as:kinase assays to detect the phosphorylation of such signal transductionmolecules, calcium mobilization assays to detect increases in calciumlevels in a cell's cytoplasm, and immunoassays such as those listedabove. Such methods are known in the art. The methods of the presentinvention can further include the step of performing a toxicity test todetermine the toxicity of a putative regulatory compound.

One embodiment of the present invention relates to a method to treat adisease involving cytokine production in an animal, comprisingregulating an MEKK/JNKK-contingent signal transduction pathway to affectcytokine production by a hematopoietic cell. Such diseases includemedical disorders and diseases in which the pathogenesis of the diseaseand/or the physiological effects of the disease might be ameliorated byregulation of cytokine production. Such diseases include, but are notlimited to, allergic diseases, anaphylaxis, diseases involving defectsin hematopoietic cells, inflammation, mast cell disorders, sepsis andcancer. According to the present invention, the term treatment can referto the regulation of the progression of a disease or the completeremoval of a disease (e.g., cure).

The present invention preferably relates to a method to treat allergicinflammation, comprising regulating cytokine production in ahematopoietic cell in an animal by regulating an MEKK/JNKK-contingentsignal transduction pathway. In one embodiment, such a hematopoieticcell is selected from a group of a mast cell, a basophil and aneosinophil, such a cell expressing FceRI.

In a preferred embodiment, a disease involving cytokine production canbe treated by administering to an animal an effective amount of acompound which interacts with a signal transduction molecule in anMEKK/JNKK-contingent signal transduction pathway in a hematopoietic cellof said animal, such that cytokine production by such a cell isregulated. In a preferred embodiment, cytokine production is inhibited.

Signal transduction molecules of an MEKK/JNKK-contingent signaltransduction pathway that can be regulated by administration of aregulatory compound include MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 andJNK2. A preferred compound to administer to an animal to regulatecytokine production by a hematopoietic cell is a compound selected fromthe group of an MEKK inhibitor, a JNKK inhibitor, and a JNK inhibitor.In a preferred embodiment, a disease involving cytokine production by ahematopoietic cell of an animal can be regulated by administering aneffective amount of a compound which inhibits PI3-K.

As used herein, an "inhibitor" of a particular signal transductionmolecule inhibits, prevents, decreases, or impedes, the normal activityof such a molecule. An inhibitor can inhibit a specific signaltransduction molecule by a means including, but not limited to: causingsuch a molecule to be degraded, binding to such a molecule such that themolecule is incapable of being activated, binding to such a moleculesuch that the molecule is unable to interact with other signaltransduction molecules, inhibiting transcription of such a molecule, andinhibiting translation of such a molecule.

As used herein, "an effective amount" of such a compound is an amount,or dose, of a regulatory compound, that when administered to an animal,is capable of regulating cytokine production by a hematopoietic cell insaid animal.

Effective doses to administer to an animal include doses administeredover time that are capable of regulating cytokine production by ahematopoietic cell in the animal. For example, a first effective dosecan comprise an amount of a regulatory compound of the present inventionthat causes a minimal change in cytokine production by a hematopoieticcell when administered to an animal. A second effective dose cancomprise a greater amount of the same compound than the first dose.Effective doses can comprise increasing concentrations of the compoundnecessary to regulate cytokine production and ameliorate a diseaseinvolving such cytokine production in an animal such that the animaldoes not have an immune response to subsequent exposure to the compound.A suitable single dose of a regulatory compound of the present inventionis a dose that is capable of substantially regulating cytokineproduction by a hematopoietic cell when administered one or more timesover a suitable time period. A preferred single dose of a regulatorycompound ranges from about 0.01 μg to about 1,000 milligrams (mg) ofsuch a compound per subject, more preferred ranges being from about 0.1μg to about 100 mg of a compound per subject, and even more preferredranges being from about 1 μg to about 10 mg of a compound per subject.

A regulatory compound of the present invention can be administered toany animal, preferably to mammals, and even more preferably to humans.Acceptable protocols to administer a regulatory compound of the presentinvention in an effective manner include individual dose size, number ofdoses, frequency of dose administration, and mode of administration.Determination of such protocols can be accomplished by those skilled inthe art depending upon a variety of variables, including the animal tobe treated and the stage of disease. Modes of delivery can include anymethod compatible with prophylactic or treatment of a disease. Modes ofdelivery include, but are not limited to, parenteral, oral, intravenous,topical administration, local administration, and ex vivo administrationto isolated hematopoietic cells.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES

For the following Examples 1-12, the materials and methods used hereinare the same throughout the examples and are therefore described indetail only upon the first appearance of such material or method.

Example 1

The following example demonstrates that JNK is activated throughaggregation of FceRI by antigen or anti-IgE in MC/9 cells.

MC/9, a mouse mast cell line, was originally derived from fetal livercells cultured in concanavalin A-conditioned medium followed by culturewith irradiated syngeneic bone marrow cells. The MC/9 mast cell line wasidentified as a source of mast cells by light microscopy and theappearance of metachromatic granules. Further characterization includesthe findings on electron microscopy and the ability to release histamineupon stimulation with A23187 or with antigen following passivesensitization with IgE. MC/9 cells express FcεRI on the cell surface.

The MC/9 murine mast cell clone was obtained from the American TypeCulture Collection (Rockville, Md.) and maintained in Dulbecco'smodified Eagle's medium (Life Technologies, Inc.) supplemented with5×10⁻⁵ M 2-mercaptoethanol (Life Technologies, Inc.), 10% fetal bovineserum (Summit Biotechnology, Ft. Collins, Colo.), and 5% conditionedmedium (rat growth factor obtained from Collaborative Biomedical(Bedford, Mass.)). Purified rat anti-mouse IgE monoclonal antibody(R35-72) was purchased from Pharmingen (San Diego, Calif.). Ovalbumin(OVA, grade V) was obtained from Sigma. Recombinant protein G-Sepharose4B was purchased from Zymed Laboratories (San Francisco, Calif.). Ananti-OVA IgE antibody-secreting hybridoma cell line was generated asdescribed. A hybridoma cell line producing monoclonal mouse IgE-specificfor 2,4,6-trinitrophenol (TNP), IGEL b4 was purchased from ATCC.

MC/9 cells (5×10⁶ /ml) were cultured with 500 ng/ml anti-OVA IgE for 2h. The cells were washed with medium three times and cultured with freshmedium for an additional 2 h. OVA dissolved in PBS or anti-IgE was addedfor the stimulation, and PBS was used as a control vehicle.

The activity of JNK was measured by monitoring the activity of aGlutathione S-transferase-c-Jun-(1-79) fusion protein. Cells (3×10⁶)were lysed in a buffer (20 mM Tris-HCl, pH 7.6, 250 mM NaCl, 3 mM EDTA,3 mM EGTA, 0.5% NP-40, 2 mM Na₃ VO₄, 1 mM dithiothreitol (DTT), 1 mMphenylmethylsulfonyl fluoride (PMSF), 20 μg/ml aprotinin, 5 μg/mlleupeptin). The lysates were mixed with GST-c-Jun-Sepharose beads androtated at 4° C. for 3 h. The beads were washed twice in lysis bufferand once in kinase assay buffer (20 mM Hepes, pH 7.5, 20 mMβ-glycerophosphate, 10 mM MgCl₂, 1 mM DTT, 50 mM Na₃ VO₄, 10 mMp-nitrophenyl phosphate). After the final wash, 40 μl of a kinase assaybuffer containing 10 μCi of γ-³² P-ATP (ICN Pharmaceuticals, Irvine,Calif.) were added per sample. The sample was incubated for 20 min at30° C. and the reaction was stopped by addition of 13 μl of 4X proteinloading buffer (188 mM Tris (pH 6.8), 30% glycerol, 6% sodium dodecylsulfate (SDS), 15% 2-mercaptoethanol, 0.4% bromophenol blue). Thesamples were boiled for 3 min, and GST-c-Jun was separated by SDS-12%polyacrylamide gel. The gel was stained with Coomassie brilliant blue,exhaustively destained, dried, and subjected to autoradiography. Thebands corresponding to GST-c-Jun were cut out of the gel, andradioactivity was determined by liquid scintillation counting.

MC/9 cells were incubated with 500 ng/ml mouse monoclonal IgE specificfor OVA (OVA-IgE) for 2 h. After washing, sensitized MC/9 cells wereincubated in the presence of 10 ng/ml to 100 g/ml OVA for 10 min.Following addition of OVA to MC/9 cells sensitized with OVA-specific IgE(OVA-IgE), JNK was significantly activated in a dose-dependent manner.10 μg/ml OVA induced maximal activation of JNK (FIG. 2A).

MC/9 cells sensitized with OVA-IgE were incubated in the presence of PBS(0 min) or 10 μg/ml OVA for 1, 3, 5, 10, 15, 20, 30, 40, or 60 min.FIGS. 2B and 2C show representative autoradiography from fourindependent experiments (2B) and fold increases in JNK activity(mean±S.D., n=4) (2C). JNK was significantly activated within 5 min, andits activation was maximal at 15-20 min after the addition of OVA (FIG.2B and 2C).

MC/9 cells were incubated with 500 ng/ml mouse monoclonal IgE specificfor TNP (TNP-IgE) or 500 ng/ml OVA-IgE for 2 h. FIG. 2D shows MC/9 cellssensitized with TNP-IgE were incubated together with PBS (control), 10μg/ml OVA (OVA), 1 μg/ml rat anti-mouse IgE monoclonal antibody(anti-IgE (1)), or 10 μg/ml anti-mouse IgE (anti-IgE (10)) for 10 min.MC/9 cells sensitized with OVA-IgE were incubated with PBS (control), 10μg/ml OVA (OVA), 10 μg/ml BSA (BSA), or 1 μg/ml anti-IgE (anti-IgE (1))for 10 min. GST, glutathione S-transferase. JNK activation by OVA wasnot induced in MC/9 cells sensitized with TNP-specific IgE (TNP-IgE) andBSA did not activate JNK in MC/9 cells sensitized with OVA-IgE.Anti-mouse IgE antibody activated JNK in both TNP-IgE andOVA-IgE-sensitized cells (FIG. 2D). Student's t test, Welch's t test, ora paired t test was used for the statistical analysis.

Example 2

The following example demonstrates that MEKK1 is activated by antigen inMC/9 cells.

Affinity-purified rabbit polyclonal anti-mouse MEK kinase 1 (MEKK1)antibody was prepared by immunizing rabbits with a recombinant fragmentof the amino-terminal domain of MEKK1.

Addition of OVA (10 μg/ml) induced MEKK1 activation in MC/9 cellssensitized with OVA-IgE. As a positive control in the kinase assay forMEKK1, cell lysates from Cos cells that transiently overexpressedfull-length MEKK1 were used.

To assay for MEKK1, MEKK1 was first immunoprecipitated by lysing 5×10⁶cells by vigorous mixing in 0.4 ml of extraction buffer (1% TritonX-100, 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 0.1%bovine serum albumin (BSA), 20 μg/ml aprotinin, 1 mMphenylmethylsulfonyl fluoride, and 2 mM Na₃ VO₄). The lysate wasincubated with the affinity-purified rabbit anti-MEKK1 antibody (1:100dilution) for 2 h at 4° C. Recombinant protein G-Sepharose 4B was addedto the lysate and incubated for an additional 30 min at 4° C. The immunecomplexes were washed twice with radioimmunoprecipitation assay buffer(10 mM sodium phosphate (pH 7.0), 150 mM NaCl, 2 mM EDTA, 1% NonidetP-40 (Nonidet P-40), 0.1% SDS, 10 μg/ml aprotinin, 1 mMphenylmethylsulfonyl fluoride, 0.1% 2-mercaptoethanol, 50 mM NaF, and200 μM Na₃ VO₄), twice with PAN buffer (10 mM PIPES pH 7.0), 100 mMNaCl, 20 μg/ml aprotinin) containing 0.5% Nonidet P-40 and once withPAN. For the in vitro kinase assay, the PAN immune complex suspensionwas incubated with catalytically inactive JNKK (JNKK-KR) and 30 μCi ofγ-³² P!ATP in 1×universal kinase buffer (20 mM PIPES (pH 7.0), 10 mMMnCl₂, and 20 μg/ml aprotinin) in a final volume of 40 μl for 30 min at30° C. MEKK1 was transiently expressed in Cos cells by usinglipofectamine (Life Technologies, Inc.), and the cell lysate was used asa positive control in the MEKK1 kinase assay. The kinase reaction wasterminated by the addition of 4×protein loading buffer, and the mixturewas boiled for 5 min, separated by SDS-10% polyacrylamide gel, andtransferred to nitrocellulose for autoradiography and immunoblotting.The kinase activity was quantified with a PhosphorImager (MolecularDynamics, Sunnyvale, Calif.). The membranes were probed using the sameanti-MEKK1 antibody with an alkaline phosphatase visualization system(Promega protoblot alkaline phosphatase system, Madison, Wis.).

MC/9 cells sensitized with OVA-IgE were incubated together with PBS (0min) or 10 μg/ml OVA for 0.5, 1, 3, 5, 10, or 20 min. The cell lysatefrom Cos cells which expressed full-length MEKK1 (Cos (MEKK1)) was usedas a positive control in the kinase assay. MEKK1 activation in MC/9cells was observed 30 s after the addition of OVA to IgE-sensitizedcells. MEKK1 activity reached maximal levels 3 min after OVA addition.MEKK1 activity was increased to 2.5-3-fold over basal activity. FIG. 3Aand 3B show a representative autoradiograph from three independentexperiments (FIG. 3A) and fold increases in MEKK1 activity (mean±S.E.,n=3) (FIG. 3B). Kinase activities decreased gradually by 10 min afteraddition of OVA. The same membrane in the kinase assay was probed withthe anti-MEKK1 antibody used for immunoprecipitation, and reactivity wasvisualized by the alkaline phosphatase system to ensure that the sameamounts of MEKK1 were present in each sample. Immunoblotting showed a98-kDa bank of MEKK1, and the density in each sample was comparable(data not shown).

Example 3

The following example demonstrates that ERK2 is phosphorylated andactivated by antigen ligation in MC/9 cells.

The mouse monoclonal anti-mouse ERK2 antibody and bovine myelin basicprotein were obtained from Upstate Biotechnology (Lake Placid, N.Y.).Goat affinity-purified polyclonal anti-ERK2 (C-14, amino acids 345-358)antibody was purchased from Santa Cruz Biotechnology (Santa Cruz,Calif.).

ERK2 activation induced by tyrosine-threonine phosphorylation wasobserved in immunoblots using anti-ERK2 antibody. After differenttreatments, 1×10⁶ cells were lysed in buffer (150 mM NaCl, 1% NonidetP-40, 0.5% deoxycholic acid sodium salt, 0.1% SDS, 50 mM Tris (pH 7.6),10 μg/ml aprotinin, 5 mg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride). Samples were electrophoresed on SDS-10% polyacrylamide gelsand proteins were transferred to nitrocellulose membranes. Membraneswere incubated overnight in blocking buffer containing 1% BSA at 4° C.The monoclonal anti-ERK2 antibody (200 μg/69 μl, Upstate Biotechnology)was added to the blocking buffer (1:1000), and blots were incubated foran additional 1 h at room temperature. The blots were washed in TBST (25mM Tris (pH 8.0), 125 mM NaCl, 0.025% Tween 20), and specific reactiveproteins were detected by an enhanced chemiluminescence method,employing a sheep anti-mouse Ig antibody linked to horseradishperoxidase (Amersham Corp.).

MC/9 cells sensitized with OVA-IgE were incubated with PBS (0) or 100pg/ml to 100 μg/ml OVA for 1 min. Cell lysates were analyzed by SDS-10%polyacrylamide gel and immunoblotting using an anti-ERK2 antibody. ERK2was phosphorylated in the presence of 1-100 μg/ml OVA (FIG. 4A). MC/9cells sensitized with OVA-IgE were incubated with PBS (0 min) or 10μg/ml OVA for 0.5, 1, 5, 10, 20, 30, or 40 min. ERK2 phosphorylation waselicited within 30 s, and a clear shift in mobility was observed at 1-20min after 10 μg/ml OVA stimulation. Phosphorylated ERK2 protein wasdecreased 30-40 min after OVA addition (FIG. 4B). MC/9 cells sensitizedwith OVA-IgE were incubated with PBS or 10 μg/ml OVA for 1, 5, 20, 40,60, or 90 min. Kinase activity of ERK2 was measured as ³² Pincorporation into myelin basic protein. ERK2 was significantlyactivated at 1 min after the addition of OVA, and its activation wasmaximal at 5-20 min. Significant activation was still observed at 90 minafter OVA stimulation (FIG. 4C).

Example 4

The following example demonstrates that wortmannin inhibits JNKactivation but not ERK2 activation.

Wortmannin was obtained from Calbiochem and stored as a 10 mM stock indimethyl sulfoxide (Me₂ SO).

Wortmannin has been shown to inhibit PI3-kinase when used atconcentrations below 1 μM. The effect of wortmannin (3 nM to 1 μM) onJNK and ERK2 activation induced by 10 μg/ml OVA stimulation inOVA-IgE-sensitized MC/9 cells was examined. MC/9 cells sensitized withOVA-IgE were incubated with 0.01% Me₂ SO (control and 0 nM) or 3-1000 nMwortmannin for 15 min. The cells were then incubated with 10 μg/ml OVAor PBS (control) for 10 min. The data are expressed as the percentage ofJNK activity detected in the presence of 10 μg/ml OVA and 0.01% Me₂ SO(FIGS. 5A and 5B) or as the percentage of ERK2 activity stimulated by 10μg/ml OVA in the presence of 0.01% Me₂ SO (FIG. 5C). (*, p<0.05;**,p>0.01.)

Wortmannin inhibited JNK activity in a dose-dependent manner. The kinaseactivity in cells treated with 100 nM wortmannin was decreased to 8% ofthat observed in the absence of treatment (FIGS. 5A and 5B). Incontrast, 100-300 nM wortmannin did not significantly inhibit ERK2activation induced by OVA (FIG. 5C).

The aggregation of FcεRI initiates diverse signal transduction pathways.In addition to the release of mast cell granule contents, these pathwayslead to late responses such as the increase in c-fos and c-junexpression and modulation of cytokine gene expression. Electrophoreticmigration and activation of ERK2 was observed in antigen-stimulated MC/9cells. Based on the present invention, it is believed that a role forERKs in mast cells is the activation of cytosolic phospholipase A2,which would result in the production of arachidonic acid derivativessuch as LTC4 and PGD2. Functionally, antigen-stimulated JNK activity inmast cells functions in the regulation of AP-1 activity and cytokinegene expression. The present invention provides the first evidence forthe regulation of the MEKK/JNKK-contingent pathway, and not the ERKpathway, in mast cell cytokine production, thus permitting geneticanalysis of the role of this pathway in mast cell biology.

Example 5

The following example demonstrates that TNF-α is generated by antigen inpassively sensitized MC/9 cells.

Recombinant mouse TNF-α, purified rat anti-mouse TNF-α monoclonalantibody (ELISA Capture), and biotinylated rabbit anti-mouse TNF-αpolyclonal antibody (ELISA Detection) were purchased from Pharmingen(San Diego, Calif.).

An ELISA for TNF-α production was performed as follows. Purified ratanti-mouse TNF-α monoclonal antibody was diluted to 2 μg/ml in coatingsolution (0.1M NaHCO₃, pH 8.2) and 50 μ was added to wells of an ELISAplate (Dynatech Laboratories). After overnight incubation at 4° C.,wells were washed twice with washing solution (0.05% Tween-20/PBS) andblocked with PBS containing 10% FCS (10% FCS/PBS ) at room temperaturefor 2 hrs. After washing twice, standards (16 pg/ml-4 ng/ml recombinantmouse TNF-α) and samples were added at 100 μl per well and incubatedovernight at 4° C. After washing four times, biotinylated rabbitanti-mouse TNF-α polyclonal antibody (1 μg/ml) was added to wells andincubated at room temperature for 45 min and wells were washed sixtimes. 2 μg/ml avidin-peroxidase was added to wells, incubated at roomtemperature for 30 min, and wells were washed eight times. ABTS(2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (30mg/ml 0.1M citric acid, pH 4.35) containing 0.03% H₂ O₂ was added at 100μl per well and color reaction was allowed to develop at roomtemperature for 30 min. A plate was read at OD 410 nm and analyzed byMycroplate Manager (Bio Rad, Hercules, Calif.).

MC/9 cells (1×10⁶ /ml) were incubated with 500 ng/ml mouse monoclonalIgE specific for OVA (OVA-IgE) for 2 h. After washing, 1×10⁶ sensitizedMC/9 cells were incubated in the presence of 10 ng/ml to 100 μg/ml OVAfor 3 h. After the incubation, the cell supernatant was harvested andTNF-α production was measured by ELISA. TNF-α production reached maximalby addition of 10 μg/ml OVA (mean±S.D., n=4). 1-100 μg/ml OVA inducedTNF-α production at 3 h after addition of OVA (FIG. 6). 3.6 ng TNF-α wasproduced from 1×10⁶ cells in the presence of 10 μg/ml OVA.

MC/9 cells sensitized with OVA-IgE (1×10⁶ /ml) were incubated in thepresence of PBS (0 h) or 10 μg/ml OVA for 0.5, 1.0, 1.5. 2.0, 2.5, 3.0,or 4.0 h. TNF-α was detected 1 h after addition of OVA and reachedmaximal at 2.5-3.0 h (mean ±S.D., n=4). TNF-α was not detected in thesupernatant at 30 min after the addition of 10 μg/ml OVA. TNF-αproduction leveled at 2.5-3 hrs after the addition of OVA (FIG. 7).

The effects of a protein synthesis inhibitor, cycloheximide, and an RNAtranscription inhibitor, actinomycin D, on TNF-α production wereexamined. 1 μg/ml cycloheximide or 1 μg/ml actinomycin D was incubatedwith cells 15 min before the addition of OVA. Both cycloheximide andactinomycin D completely blocked TNF-α production 3 h after stimulation(less than 30 pg per million cells) (data not shown).

The aggregation of FcεRI on mast cells is essential for the induction ofallergic inflammation. Following aggregation, mast cells secrete avariety of preformed chemical mediators, such as histamine, and newlysynthesized arachidonic acid derivatives. In addition to thesebiologically active substances, aggregation of FcεRI on mast cells leadsto the production of cytokines and chemokines such as IL-3, IL-5, IL-6,TNF-α, GM-CSF, and MIP-1α. Among these cytokines, TNF-α, is produced inlarge amounts in mast cell lines. Both mouse bone marrow-derived mastcells and human cultured mast cells also produce TNF-α. Therefore, TNF-αis likely to be involved in allergic inflammation initiated by mast cellactivation. TNF-α is a multifunctional cytokine which has effects ininflammation. Like other cytokines, TNF-α is newly synthesized followingthe aggregation of FcεRI on mast cells. Stimulation via the FcεRImarkedly increases the levels of TNF-α mRNA in BMMC and some mast celllines. The TNF-α gene shows the characteristics of an immediate-earlygene in activated mast cells. It is strongly induced within 30 min inantigen-stimulated mast cells. MC/9, which does not have preformedTNF-α, produces TNF-α following the aggregation of FcεRI. The presentinventors have shown herein that cycloheximide and actinomycin Dcompletely inhibited TNF-α production induced by FcεRI aggregation,demonstrating that TNF-α is synthesized de novo following activation inMC/9 cells.

Example 6

The following example illustrates that p38 is activated by antigen inMC/9 cells.

MC/9 cells sensitized with OVA-IgE were incubated in the presence of PBS(0 min) or 10 μg/ml OVA for 1, 5, 15, 30, or 60 min. Toimmunoprecipitate p38 kinase, 3×10⁶ cells were lysed by vigorous mixingin 0.4 ml of extraction buffer (1% Triton X-100, 10 mM Tris-HCl (pH7.4), 5 mM EDTA, 50 mM NaCl, 50 mM MaF, 0.1% bovine serum albumin (BSA),20 μg/ml aprotinin, 1 mM PMSF, and 2 mM Na₃ VO₄). The lysate wasincubated with the rabbit anti-serum raised against the COOH-terminalpeptide sequence of p38 (1:400 dilution) for 2 h at 4° C. Recombinantprotein G sepharose 4B was added to the lysate and incubated for anadditional 1 h at 4° C. The immunoprecipitates were washed once withextraction buffer, twice with PAN buffer (10 mM piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES, pH 7.0), 100 mM NaCl, 20 μg/mlaprotinin). For the in vitro kinase assay, the immunoprecipitates weresuspended in 25 μl of assay buffer (25 mM Hepes, pH 7.4, 25 mMβ-glycerophosphate, 25 mM NaCl₂, 2 mM dithiothreitol, 0.1 mM Na₃ VO₄)containing a recombinant NH₂ terminal fragment of ATF-2 (20-50 ng) as asubstrate and 5 μCi γ³² P! ATP. The kinase reaction was terminated bythe addition of 4×protein loading buffer, and the mixture was boiled for5 min and separated by SDS-12% polyacrylamide gel. The gel was fixedwith 5% acetic acid and 10% methanol solution, dried, and subjected toautoradiography. The kinase activity was quantified with aPhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).

Following addition of OVA to MC/9 cells sensitized with OVA-IgE, p38 wassignificantly activated. OVA induced p38 activation in a dose-dependentmanner (data not shown). p38 was significantly activated at 1 min andits activation was maximal (about 12-fold increase) at 5 min after theaddition of OVA. p38 activities decreased gradually after 15-60 min(FIG. 8).

Example 1 demonstrates that JNK is strongly activated in mast cellsfollowing aggregation of FcεRI. This example demonstrates that anothermember of MAP kinase family, p38, the osmotic imbalance responsivekinase similar to the yeast Hog1 enzyme (#Lin) is also activated in mastcells by aggregation of FcεRI. p38, like JNK, is also activated bytreatment of cells with pro-inflammatory cytokines and environmentalstress such as extracellular changes in osmolarity. Its activationdepends on dual phosphorylation on threonine-180 and tyrosine-182.

Example 7

The following example demonstrates that wortmannin inhibits both p38 MAPkinase activation and TNF-α production.

MC/9 cells sensitized with OVA-IgE(3×10⁶) were preincubated with either1-1000 nM wortmannin or a control (0.01% DMSO) for 15 min and stimulatedfor 5 min by addition of 10 μg/ml OVA. Wortmannin inhibited p38 MAPKactivity in a dose-dependent manner. 100 nM-1 μM wortmanninsignificantly inhibited p38 MAPK activation (50-60% decrease in kinaseactivities) (FIG. 9). The data are expressed as the percentage of p38activity detected in the presence of 10 μg/ml OVA and 0.01% DMSO (*,p<0.05). However, the inhibitory effects of wortmannin on p38 MAP kinaseactivation in stimulated MC/9 cells were not as strong as the inhibitoryeffects of wortmannin on JNK activation as shown in Example 4.

The effect of wortmannin on TNF-α production in MC/9 cells was alsoexamined. MC/9 cells sensitized with OVA-IgE (1×10⁶ /ml) were incubatedwith 0.01% DMSO (control and 0 nM) or 1-1000 nM wortmannin for 15 min.The cells were then incubated with 10 μg/ml OVA for 3 h. TNF-α in themedium was measured by ELISA. 1 nM-1 μM wortmannin inhibited TNF-αproduction in a dose-dependent manner (mean±S.D., n=4,*, p<0.05;**,p<0.01). TNF-α production was decreased by 77% in the presence of 100 nMwortmannin (FIG. 10).

This example shows that p38 activation, like JNK activation, issignificantly inhibited by the PI3-kinase inhibitor, wortmannin,treatment. PI3-kinase is an enzyme important in intracellulartrafficking, actin polymerization, and growth factor signaling.PI3-kinase is activated following aggregation of FcεRI in a ratbasophilic leukemia cell line, RBL-2H3. The inhibitory effects ofwortmannin on JNK and p38 activation were observed in antigen-stimulatedmouse bone marrow derived mast cells as well as MC/9 cells (data notshown). Wortmannin also inhibited TNF-α production of antigen-stimulatedMC/9 cells in a dose-dependent manner. The concentrations of wortmanninwhich inhibited TNF-α production were similar to concentrations at whichwortmannin inhibits PI3-kinase. These results indicate that inhibitionof PI3-kinase by wortmannin decreases JNK and p38 activation followingFcεRI aggregation and that activation of p38 can enhance the effects ofthe MEKK/JNKK-contingent pathway of the present invention on TNF-αproduction in antigen-stimulated mast cells.

Example 8

The following example illustrates that the MEK inhibitor, PD 098059,inhibits ERK2 activation, but does not inhibit TNF-α production, JNKactivation, or p38 activation in MC/9 cells.

MEK inhibitor, PD#098059, was kindly provided by Dr. David Dudley(Warner Lambert Company, Ann Arbor, Mich.) and stocked 100 mM in DMSO.PD 098059 is a noncompetitive inhibitor of MAP kinase kinase (MEK). Itexerts its effect by binding to the inactive form of MEK1.

MC/9 cells sensitized with OVA-IgE (1×10⁶ /ml) were incubated with 0.1%DMSO (control and 0 nM) or 3-30 μM PD 098059 for 1 h and the reactionwas stopped by centrifugation at 5 min after the stimulation. The cellswere then incubated with 10 μg/ml OVA or PBS (control) for 5 min. Kinaseactivity of ERK2 was measured as ³² P incorporation into myelin basicprotein (MBP).

1×10⁶ cells were lysed in buffer (150 mM NaCl, 1% NP40, 0.5% deoxycholicacid sodium salt, 0.1% SDS, 50 mM Tris (pH 7.6), 10 μg/ml aprotinin, 5mg/ml leupeptin, 1 mM PMSF). Samples were electrophoresed on SDS-10%polyacrylamide gels and proteins were transferred to nitrocellulosemembranes. Membranes were incubated overnight in blocking buffercontaining 1% BSA at 4° C. The monoclonal anti-ERK 2 antibody (200 μg/69μl, Upstate Biotechnology) was added to the blocking buffer (1:1000) andblots were incubated for an additional 1 h at room temperature. Theblots were washed in TBST (25 mM Tris, pH 8.0, 125 mM NaCl, 0.025% Tween20) and specific reactive proteins were detected by an enhancedchemiluminescence method, employing a sheep anti-mouse Ig antibodylinked to horseradish peroxidase (Amersham, Arlington Heights, Ill.).

In vitro kinase assay of ERK2 was carried out as described above withsome minor modifications. 3 10⁶ cells were lysed by vigorous mixing in0.4 ml of lysis buffer (20 mM Tris-HCl (pH 8.0), 1% Triton X-100, 10%glycerol, 137 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 50 mM NaF, 1 mM Na₃ VO₄,1 mM PMSF, 5 μg/ml leupeptin, and 10 μg/ml aprotinin). The lysate wasincubated with anti-ERK2 antibody (2 μg/ml) for 2 hrs at 4° C.Recombinant protein G sepharose 4B was added to the lysate and incubatedfor an additional 1 hr at 4° C. The immune complexes were washed threetimes with lysis buffer, and once with kinase buffer (30 mM Tris-HCl (pH8.0), 20 mM MgCl₂, 2 mM MnCl₂). For the in vitro kinase assay, theimmune complex suspension was incubated with 9 μg myelin basic protein(MBP) and 10 μCi of γ-³² P! ATP in kinase buffer in a final volume of 30μl for 30 min at 30° C. The reaction was stopped by an additional 10 μlof 4×protein loading buffer. After the samples were boiled for 3 min,they were separated by SDS-12% polyacrylamide gel and stained withCoomassie brilliant blue, exhaustively destained, dried, and subjectedto autoradiography. The bands corresponding to MBP were cut out of thegel, and radioactivity was determined by liquid scintillation counting.

10-30 μM PD 098059 significantly inhibited ERK2 activities in MC/9cells. OVA-induced ERK2 activity was decreased 65% in the presence of 30μM PD 098059 (FIG. 11). The data are expressed as the percentage of ERK2activity detected in the presence of 10 μg/ml OVA and 0.1% DMSO (*,p<0.05;**, p<0.01). However, PD 098059 did not inhibit JNK or p38activation (data not shown).

MC/9 cells sensitized with OVA-IgE (1×10⁶ /ml) were incubated with 0.1%DMSO or 30 μM PD 098059 for 1 h. The cells were then incubated with 10μg/ml OVA for 3 h. TNF-α in the medium was measured by ELISA. 30 μM PD098059 did not affect TNF-α production (mean±S.D., n=4, NS, nosignificance). In contrast to the effect seen with wortmannin in example7, PD 098059 did not affect on TNF-α production at 3 h after addition ofOVA (FIG. 12).

Cytokine production in mast cells via FcεRI is mediated by thephosphoinositide hydrolysis, an increase in intracellular calcium, andprotein kinase C (PKC) activation because such production can be inducedby calcium ionophore or the protein kinase C activator, PMA. MC/9 cellsalso produce cytokines including IL-2, IL-3, IL-4, and GM-CSF, afterstimulation with PMA (phorbol myristate acetate) plus the calciumionophore A23187. Prior to the present invention, it was thought thatthe Ras-dependent ERK signal transduction pathway was the intermediatein the transduction pathway leading to increases in gene transcriptionand proliferation in mast cells. ERKs phosphorylate specifictranscription factors including members of the Ets family such as Elk-1.MEK inhibitor, PD 098059, selectively blocks the activation of MEK1 byRaf or MEKK in vitro. It exerts its effect by binding to the inactiveform of MEK1, inhibiting both the phosphorylation and activation of ERK.

The present inventors have shown herein that PD 098059 stronglyinhibited both ERK2 activity and phosphorylation induced by theaggregation of FcεRI in MC/9 cells. However, it did not inhibit TNF-αproduction, JNK activation or p38 activation. On the contrary, PD 098059enhanced OVA-induced promoter activity of TNF-α although it did notenhance the TNF-α level in the medium. This demonstrates that ERK2activation is not required for TNF-α production in mast cells. Withoutbeing bound by theory, the present inventors believe that ERK2 might bea negative regulator of transcription of TNF-α since inhibition of ERK2activation enhanced activity of the TNF-α promoter in antigen-stimulatedMC/9 cells.

Example 9

The following example demonstrates that wortmannin, but not PD 098059,inhibits TNF-α promoter activity in MC/9 cells stimulated by FcεRIaggregation.

The pXP1 plasmid containing full length of the human TNF-α promoter justupstream of the luciferase gene, designated pTNF(-1311)Luc, was providedby Dr. James S. Economou (Division of Surgical Oncology, UCLA School ofMedicine, Los Angels, Calif.). pTNF(-01311)Luc was transfected into MC/9by the DEAE-dextran method. 2×10⁶ cells were washed once with 1×TBS (25mM Tris, 137 mM NaCl, 5 mM KCl, 0.5 mM Na₂ HPO₄, 0.49 mM MgCl₂, 0.68 mMCaCl₂, pH 7.5). Cells were suspended with 0.4 ml of 500 μgDEAE-dextran/4 μgDNA mixture and incubated at room temperature for 30min. After washed with 1×TBS, cells were suspended with 10 ml of culturemedium and plated on culture dish. After 24 h of the transfection, cellswere passively sensitized with OVA-IgE and incubated with 10 μg/ml OVAfor additional 15 h. In the experiment for cotransfection, 10 μgpCMV5MEKK_(COOH), expression plasmid encoding MEKK_(COOH), a truncatedactivated form of MEKK1 or 10 μg pCMV5, control empty plasmid, wastransfected with 4 μg pTNF(-1311)Luc and transfected cells wereharvested after 24 h of the cotransfection. For in vitro kinase assay, 4μg or 10 μg pCMV5MEKK_(COOH), or equivalent amount of control emptyplasmid was transfected similarly and cells were harvested for in vitrokinase assay 24 h after the transfection.

Luciferase activity was measured to measure TNF-α promoter activity.Cell pellets were lysed in 200 μl of a buffer containing 25 mM Tris, pH7.8, 2 mM EDTA, 2 mM dithiothreitol, 10% glycerol, 1% Triton X-100. 30μl of the lysate was mixed with the same volume of Luciferase AssaySubstrate containing beetle luciferin as a substrate (Promega, MadisonWis.), and chemiluminescence was measured for 30 sec as relative lightunits by a luminometer (Monolight 2010, Analytical LuminescenceLaboratory, San Diego, Calif.).

MC/9 cells transiently transfected by pTNF(-1311)Luc were passivelysensitized with OVA-IgE, and incubated for an additional 15 h with 10μg/ml OVA or PBS in the presence of 0.01% DMSO or 100 nM wortmannin.Similarly, cells were incubated with 10 μg/ml OVA or PBS in the presenceof 0.1% DMSO or 30 μM PD 098059. Luciferase activity in cell lysates wasmeasured as relative light units (RLU) and standardized by control RLU(mean±S.D., N=4, p<0.01). OVA addition elicited a 5-6 fold induction ofluciferase activity in the cells. 100 nM wortmannin significantlyinhibited luciferase activity induced by addition of OVA (40% decreasein luciferase activity) (FIG. 13). In contrast to wortmannin, PD 098059enhanced OVA-induced luciferase activity (FIG. 14). Therefore, the PI3-Kinhibitor, wortmannin, inhibited TNF-a promoter activity in MC/9 cells,whereas the MEK inhibitor, PD 098059, enhanced TNF-a promoter activity.

Example 10

The following example demonstrates that overexpression of MEKK1 by MC/9cells greatly enhances the activity of JNK and TNF-α promoter, weaklyenhances p38 activity and ERK2 activity in antigen-activated MC/9 cells.4 or 10 μg of pCMV5MEKK_(COOH) (MEKK1(4) or MEKK1 (10)) or equivalentamount of pCMV5 empty plasmid (pCMV5(4) or pCMV5(10)) was transfectedinto MC/9 cells. Cells treated with only DEAE-dextran were used as acontrol. JNK activities (FIG. 15), p38 activities (FIG. 16), and ERK2activities (data not shown) were measured at 24 h after thetransfection. FIGS. 15-16 graphically show representativeautoradiographs from each three of independent experiments. Kinaseactivities were standarized by control activity and expressed asfold-activation (mean±S.D., n=3,*, p<0.05;**, p<0.01).

JNK activity in the MC/9 cells transiently transfected bypCMV5MEKK_(COOH) was strongly increased compared with the cellstransfected by pCMV5 empty plasmid or treated with DEAE-dextran(Pharmacia Biotech, Uppsala, Sweden) alone. More than 11-15 foldincreases of JNK activity was observed in pCMV5MEKK_(COOH) -transfectedcells compared with pCMV5 empty plasmid-transfected cells (FIG. 15). Incontrast, p38 activity was increased to a much lesser degree by thetransfection of pCMV5MEKK_(COOH) (FIG. 16). ERK2 was also activated bypCMV5MEKK_(COOH) transfection. However, the degree of ERK2 activationwas less than that of JNK (data not shown).

10 μg of pCMV5MEKK_(COOH) (MEKK1) or 10 μg of pCMV5 empty plasmid(pCMV5) was transfected into MC/9 cells with 4 μg of pTNF(-1311)Luc.Luciferase activities were measured as relative light units at 24 hafter the transfection.

Cotransfection of pCMV5MEKK_(COOH) and pTNF(-1311)Luc elicited a1000-fold induction of luciferase activities compared withcotransfection of pCMV5 empty plasmid and pTNF(-1311)Luc, demonstratingthat overexpression of MEKK1 results in an increase in the level ofTNF-α promoter activity (FIG. 17).

A protein kinase cascade leading to activation of JNK is dependent onMEK kinase 1 (MEKK1). MEKK1 was identified as MEK-activating kinaseunrelated to Raf-1. MEKK1 is activated in both a Ras-dependent and-independent manner. Examples 3A and 3B show, for the first time, thatMEKK1 is also activated following the aggregation of FcεRI in MC/9cells. Furthermore, the present inventors show herein thatoverexpression of MEKK1 strongly induces JNK activation and only weaklyinduces ERK activation.

JNKK activates both JNK and p38. As shown herein, however, JNK wasstrongly activated, but the activation of p38 was poor, in pCMV5_(COOH)-transfected MC/9 cells. overexpression of MEKK1 induced by transfectionof pCMV5MEKK_(COOH) enhanced the activity of TNF-α promoter strongly inMC/9 cells. Luciferase activity in pCMV5MEKK_(COOH) -transfected cellswas 1000-fold higher than that in the cells transfected by controlvector. These results demonstrate that activation of TNF-α promoterinduced by MEKK1 overexpression is caused by JNK activation. Thus, MEKK1and JNKK activation leading to JNK activation is directly involved inthe gene transcription of TNF-α in antigen-stimulated becauseaggregation of FcεRI induces the activation of both MEKK1 and TNF-αpromoter. Taken together, the above examples show that theMEKK/JNKK-contingent pathway is the primary signal transduction pathwayleading to TNF-α production by mast cells, but other wortmanninsensitive pathways such as p38 can enhance such transcription of TNF-αin antigen-stimulated mast cells.

Example 11

The following example illustrates that cross-linking of c-kit on MC/9cells synergizes with FceRI aggregation to greatly enhance both JNKactivation and TNF-a production. FIG. 18 shows that JNK activity isgreatly enhanced in MC/9 cells activated by FceRI aggregation.Cross-linking of c-kit by c-kit ligand in the absence of FceRIaggregation only weakly activates JNK. However, when both c-kit andFceRI are cross-linked, JNK activation is enhanced almost 4 fold abovethe level achieved with FceRI aggregation alone.

FIG. 19 shows the results of a similar experiment in which TNF-aproduction was measured. Like JNK, TNF-a production was greatly enhancedwhen both c-kit and FceRI were cross-linked as compared to cross-linkingeither receptor alone.

Together, these experiments further demonstrate that activation ofsignal transduction by c-kit synergizes with FceRI aggregation toactivate the MEKK/JNKK-contingent signal transduction pathway of thepresent invention.

Example 12

This example demonstrates that aggregation of FceRI on MC/9 cellsactivates the transcription factor NFkB. FIG. 20 shows thatcross-linking of FceRI on MC/9 cells activates the transcription factorNFkB. Since NFkB is known to interact with various cytokine promoters toinduce cytokine production, this example illustrates that FceRIaggregation, which has been shown herein to induce theMEKK/JNKK-contingent pathway of the present invention, can activatetranscription factors involved in cytokine production.

Another transcription factor, the nuclear factor of activated T cells(NF-AT) is essential for transcription of the IL-2 gene in activated Tcells. Without being bound by theory, it is believed that NF-AT may beone in a family of related transcription factors that regulate thetranscription of cytokine genes in mast cells as well as T cells becauseaggregation of the FcεRI induces NF-AT DNA binding activity in rat mastcells. NF-AT binding motifs are present in the promoter region of TNF-αgenes as well as in the IL-2, IL-3, IL-4, and GM-CSF promoters.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

What is claimed is:
 1. A method to treat allergic inflammation in ahuman, comprising administering to said human an effective amount of acompound which directly interacts with a signal transduction molecule inan MEKK/JNKK-contingent signal transduction pathway selected from thegroup consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2,said interaction resulting in the inhibition of cytokine production suchthat allergic inflammation is thereby treated.
 2. A method of using ahematopoietic cell to screen putative regulatory compounds for theirability to regulate cytokine production in said cellcomprising:providing a hematopoietic cell having an MEKK/JNKK-contingentsignal transduction pathway; contacting said cell with a putativeregulatory compound; and determining whether said putative regulatorycompound is capable of modulating the amount of cytokine produced bysaid cell, wherein said cell lacks an ERK-dependent signal transductionpathway.